【作者】 张渊；

【导师】 邱宗荫；

【作者基本信息】 重庆医科大学 ， 药理学， 2011， 博士

【摘要】 目的以非小细胞肺癌(NSCLC)临床患者尿液中可溶性蛋白质组和exosomes蛋白质组为研究对象,筛选可能存在于尿液中的与非小细胞肺癌相关的潜在生物标志物,为临床实施NSCLC个体化治疗和早期诊断及预后提供新的理论基础和技术策略。策略与方法1.非小细胞肺癌患者与健康对照者尿液中可溶性蛋白质组的比较分析:分别收集3例非小细胞肺癌临床患者的尿样与3例健康对照者的混合尿样,通过1D SDS-PAGE对其可溶性蛋白质组进行分离,用Quantity One软件分析电泳结果,筛选差异蛋白条带。候选差异蛋白条带经胰酶胶内酶解,HPLC-CHIP-MS/MS分析,得到的数据以Spectrum Mill Proteomics Workbench Rev A.03.03.078 (Agilent Technologies)自动分析MS和MS/MS数据,搜索UniProtKB/SWISS-PORT, Homo Sapiens(Human)数据库,并对差异蛋白质进行生物信息学分析。2.候选差异蛋白AACT的western blot和免疫组织化学验证:分别提取3例非小细胞肺癌临床患者与3例正常对照者尿样中可溶性蛋白质组,通过western blot对尿液中AACT的表达水平进行分析;收集20例非小细胞肺癌组织标本和20例配对癌旁正常肺组织标本,通过免疫组织化学技术对AACT在肺癌组织内表达情况进行分析。3.尿液exosomes中低丰度蛋白质组的分析:以超速离心法分离人尿液中的exosomes,通过透射电镜观察其形态,通过免疫电镜观察exosomes上AQP-2的表达情况。通过组合肽配体库技术(CPLL)去除尿液exosomes中的高丰度蛋白,将得到的低丰度蛋白质经胰酶酶解后通过OFFGEL电泳进行分级,采用HPLC-CHIP-MS/MS对分离后的样品进行蛋白质鉴定,最后对鉴定出的蛋白质进行基因本体(Gene ontology, GO)和相关生物信息学分析。4.非小细胞肺癌患者与健康对照者尿液中exosomes蛋白质组的比较分析:分别收集10例健康志愿者的混合尿样和8例NSCLC初诊未进行化疗患者的尿样,以超速离心法分离尿液中的exosomes,通过1D SDS-PAGE对exosomes蛋白质组进行分离,用Quantity One软件分析电泳结果,筛选差异蛋白条带。候选差异蛋白条带经胰酶胶内酶解后进行HPLC-CHIP-MS/MS分析,得到的数据以Spectrum Mill Proteomics Workbench Rev A.03.03.078 (Agilent Technologies)自动分析MS和MS/MS数据,搜索UniProtKB/SWISS-PORT, Homo Sapiens(Human)数据库,并对鉴定出的差异蛋白质进行生物信息学分析。5.候选差异蛋白LRG1的western blot和免疫组织化学验证:分别提取6例非小细胞肺癌临床患者与6例健康对照者尿样的exosomes蛋白质组,通过western blot对exosomes中的LRG1蛋白表达水平进行分析;收集20例非小细胞肺癌组织标本和10例配对癌旁正常肺组织标本,通过免疫组织化学技术对LRG1在肺癌组织内的表达情况进行分析。结果1.通过对非小细胞肺癌患者与健康对照者尿液中可溶性蛋白质组的比较分析,从差异条带中共鉴定出40种蛋白质,值得关注的是,其中8种蛋白质与非小细胞肺癌相关:半乳凝素-3-结合蛋白、凝聚素、锌-a2-糖蛋白、α-1-抗胰凝乳蛋白酶、α-1-抗胰蛋白酶、激肽释放酶、凝溶胶蛋白、富亮氨酸α-2-糖蛋白。2.western blot结果表明:非小细胞肺癌患者尿液中AACT的表达水平明显高于健康对照者,与质谱结果相符合。免疫组织化学染色结果表明:AACT主要定位于细胞浆,其在非小细胞肺癌组织中的阳性表达显著高于癌旁正常肺组织。3.通过透射电镜对分离到的尿液exosomes进行观察表明:尿液exosomes为呈明显异质性的圆形或椭圆形小囊泡,直径约30～100nm,有完整的包膜,内为低电子密度物质。在标记金颗粒的AQP-2作用于exosomes后,许多exosomes小体上有金颗粒附着,与文献报道相符。4.通过1D SDS-PAGE对CPLL处理前后的exosomes蛋白质组分析表明:尿液exosomes中含有种类丰富的蛋白质,经CPLL处理后,高丰度蛋白条带明显减弱,且出现了许多清晰的低丰度蛋白条带。5.将得到的exosomes低丰度蛋白质经胰酶酶解后通过OFFGEL电泳进行分级,采用HPLC-CHIP-MS/MS对分离后的样品进行蛋白质鉴定,共鉴定出512种非冗余蛋白质,其中374种在之前的尿蛋白质组研究中未见报道。GO分析结果显示,其中55.36%为可溶性蛋白,44.93%为膜蛋白。6.通过比较非小细胞肺癌患者与健康对照者尿液中exosomes蛋白质组的差异表达情况,从45kD～35kD条带中共鉴定出18种蛋白质,其中,11种仅来源于NSCLC患者组,4种仅来源于健康对照者组,3种同时来源于健康对照者组和NSCLC患者组。7.western blot结果表明:非小细胞肺癌患者尿exosomes中LRG1的表达水平明显高于正常对照者,与质谱结果相符合。免疫组织化学染色结果表明:LRG1主要定位于细胞浆,其在非小细胞肺癌组织中的阳性表达显著高于癌旁正常肺组织。结论1.本实验中建立的一维电泳结合二维纳流液相色谱-串联质谱的分析方法可有效地用于比较蛋白质组研究。2.AACT在NSCLC患者尿液中的高表达可能来源于肺癌组织,可作为NSCLC在尿液中检测的潜在生物标志物。3.CPLL技术能有效地去除exosomes蛋白质组中的高丰度蛋白质,使低丰度蛋白质得到有效富集。4.尿液exosomes中含有种类异常丰富的低丰度蛋白质,本研究的结果丰富了我们对尿液exosomes蛋白质组的认识。5.LRG1在NSCLC患者尿液exosomes中的高表达可能与非小细胞肺癌相关,可能作为其在尿液中检测的潜在生物标志物。更多还原

【Abstract】 ObjectiveIn the present study, we aimed to screen for candidate NSCLC-related biomarkers in urine by researching on the urinary soluble and exosomal proteome of non-small cell lung cancer (NSCLC) patients, and to lay new theoretical basis for the diagnosis, prognosis and personal therapy of NSCLC patients and provide new technological strategy.Methods1. Comparative analysis of soluble urinary proteins of normal individuals and NSCLC patients: Urinary proteomes between 3 normal persons and 3 NSCLC patients were compared by 1D SDS-PAGE. After 1D SDS-PAGE analysis, Agilent nanoHPLC-chip-MS/MS ION TRAP 6330 was used to characterize differentially expressed proteins in the bands. The MS/MS data was then automatically searched against UniProtKB/Swiss-Prot protein database by Spectrum Mill Proteomics Workbench Rev A.03.03.078 software. 2. Validation of putative urinary biomarker by western blot and immunohistochemistry staining : To validate the LC-MS/MS data, we analyzed the levels of AACT in three healthy and NSCLC urine samples by western blot;To verify the expression level of AACT in lung tissues, immunohistochemistry staining was performed on paraffin-embedded tissue sections that contained both tumor tissue and adjacent non-tumor lung tissue from 20 NSCLC patients.3. Comprehensive analysis of low abundance proteome in human urinary exosomes:We employed differential ultracentrifugation to purify urinary exosomes. To verify whether the sediments separated from normal human urine were exosomes, the vesicles were concentrated and visualized by immunogold electron microscopy using antibodies reacting with water channel aquaporin-2 (AQP2). To comprehensively explore the low abundance proteome, combinatorial peptide ligand libraries, combined with peptide OFFGEL electrophoresis were employed for the enrichment and separation of relatively low abundant proteins in urinary exosomes. After peptide OFFGEL electrophoresis, separated peptides were analyzed by nanoHPLC-chip-MS/MS. Gene ontology (GO) analysis was also employed to analyze the biological process, molecular function, and cellular component of the low abundance proteome of urinary exosomes.4. Comparative analysis of urinary exosomal proteins of normal individuals and NSCLC patients: Urinary exosomal proteomes were compared by 1D SDS-PAGE between 10 normal persons and 8 NSCLC patients. After 1D SDS-PAGE analysis, Agilent nanoHPLC-chip-MS/MS ION TRAP 6330 was utilized to characterize differentially expressed proteins in the bands. Peptide and protein identifications were run automatically with the Spectrum Mill Proteomics Workbench Rev A.03.03.078 software.5. Validation of candidate urinary exosomal biomarker by western blot and immunohistochemistry staining:To validate the LC-MS/MS data, we analyzed the levels of LRG1 in six healthy and NSCLC urinary exosomes by Western blot;To verify the expression level of LRG1 in lung tissues, immunohistochemistry staining was performed on paraffin-embedded tissue sections that contained 20 tumor tissue and 10 adjacent non-tumor lung tissue from NSCLC patients.Results1. A total of 40 unique proteins were identified by comparative analysis of urinary soluable proteins from normal individuals and NSCLC patients. Among these proteins, eight proteins were related with NSCLC, they were: CLU, KLK1, Gelsolin, LRG1, Galectin-3-binding protein, ZAG, AACT, and AAT.2. The level of AACT was higher on the whole in the urine from three NSCLC patients compared to samples from normal healthy individuals by western blot analysis. The result of immunohistochemistry staining showed significantly increased AACT level in lung cancer tissues compared with normal lung tissues.3. Purified exosomes are relatively round vesicles ranging from approximately 30–100 nm in diameter. The expression of AQP2 on the vesicles also argues positively for the presence of exosomes, as has been previously reported in human urine samples.4. 1D SDS-PAGE analysis of all fractions under reducing conditions revealed different patterns of proteins. After treatment with CPLL, very different patterns were obtained for the column eluates, which showed a large decrease of the intense bands, and the apparition of many new protein bands.5. The combinatorial peptide ligand library treated urinary exosomes were predigested and fractionated by peptide OFFGEL electrophoresis and characterized by mass detection of HPLC-CHIP-MS/MS analysis. By using this strategy, 512 non-redundant proteins were identified from the human urinary exosomes, and 374 proteins had not been reported. GO analysis showed that 55.36% of identified proteins were soluble proteins, and 44.93% were membrane proteins.6. A total of 18 proteins were identified in the 45kD～35kD band by comparative analysis of urinary exosomal proteins from normal individuals and NSCLC patients. Among them, 4 were only from normal individuals, 11 were only from NSCLC patients, and the other 3 were from both normal individuals and NSCLC patients.7. The result of western blot showed that the level of LRG1 was higher on the whole in the urinary exosomes from NSCLC patients compared to samples from healthy individuals. The result of immunohistochemistry staining showed significantly increased AACT level in lung cancer compared with normal lung tissue.Conclusions1. In the present research, using a comparative proteomic discovery approach combined 1D SDS-PAGE and nano-HPLC-chip-MS/MS, we effectively identified and validated candidate urinary biomarkers for objective and non-invasive diagnosis of NSCLC.2. The higher expression level ofAACT in urine may be associated with lung tissue of NSCLC patients, and the results suggested that LRG1 may be a candidate biomarker for non-invasive diagnosis of NSCLC in urine.3. The CPLL technology can effectively remove the abundant proteins and enrich the relatively low abundance proteome of exosmes.4. The urinary exosomal proteome contains exceedingly rich low abundance proteins, and the research result contributes a lot to our understanding of the urinary exosomal proteome.5. The higher expression level of LRG1 in urinary exosomes may be associated with lung tissue of NSCLC patients, and the results suggested that LRG1 may be a candidate biomarker for non-invasive diagnosis of NSCLC in urine.更多还原